This invention relates to a method of detecting degradation of RNA, and particularly to that by separating and detecting rRNA in microchannel electrophoresis.
mRNA is widely used in biosciences for cDNA cloning, construction of expressed sequence tag (EST) databases, gene expression analysis, etc. In diagnostic molecular pathology, mRNA is also used frequently to detect or quantitate the levels of specific gene expression, such as bcr-ab1 translocation of Philadelphia chromosome in leukemia (Bortolin, et al., "Quantitative RT-PCR combined with time-resolved fluorometry for determination of BCR-ABL mRNA", Clin Chem 1996; 42:1924-9), point mutation of p53 gene in breast cancer (Lundeberg, et al., "Assessment of sequence-based p53 gene analysis in human breast cancer: messenger RNA in comparison with genomic DNA targets", Clin Chem 1998; 44:455-62), prostate-specific antigen transcript for micrometastasis of prostate cancer (Gala, et al., "Expression of prostate-specific antigen and prostate-specific membrane antigen transcripts in blood cells: Implications for the detection of hematogenous prostate cells and standardization", Clin Chem 1998; 44:472-81), etc. Many technologies and commercial kits are available for the purification of mRNA or total RNA from cells and tissues. However, the quality of mRNA is major concern among scientists because of its extreme instability against contaminated RNases. This is particularly important when the object is to isolate full-length DNA or to analyze gene expression, quantitatively as clinical diagnostics. The ratio of optical absorbency at 260 nm and 280 nm is the most common technique for the quality assessment of RNA. However, this provides information only as to whether or not proteins are contaminated in samples. Although Northern blot or reverse transcription-polymerase chain reaction (RT-PCR) is used to amplify housekeeping genes, the existence of PCR products of these genes in samples does not guarantee that RNA is entirely intact. An assay has been developed to determine the amount of total mRNA by capturing mRNA onto oligo(dT)-immobilized microplates followed by Yoyo-1-fluorescence measurement (Miura, et al., "Fluorometric determination of total mRNA with oligo(dT) immobilized on microtiter plates", Clin Chem 1996; 42:1758-64) or calorimetric detection of incorporated biotin-mononucleotides during cDNA synthesis on the microplate (Tominaga, et al., "Colorimetric ELISA measurement of specific mRNA on immobilized-oligonucleotide-coated microtiter plates by reverse transcription with biotinylated mononucleotides", Clin Chem 1996;42:1750-57). However, the quantity of mRNA does not mean that mRNA are free from degradation, because partially digested mRNA may be captured by oligo(dT). When purified mRNA is separated by agarose gel electrophoresis and stained with ethidium bromide, one can see the smear of mRNA. If the smear is distributed to a large molecular weight region, mRNA is considered to be of good quality. However, this assay is not quantitative. Therefore, there is no suitable procedure available for the analysis of the quality of mRNA.
Interestingly, a gold standard method exists for total RNA by agarose gel electrophoresis to identify 2-3 major bands, 28s, 18s, and 7S rRNA/(Neumaier, et al., "Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics", Clin Chem 1998;44:12-26). If these bands disappear, RNA of the samples is considered as useless because RNA is digested by contaminated RNases during the purification procedure. Although agarose gel electrophoresis is easy to accomplish, one should take extra care against RNase contamination in an electrophoresis chamber, loading buffer, separation buffer, agarose gel, etc. Moreover, because of low sensitivity of ethidium bromide against RNA, a relatively large quantity of purified RNA is consumed by agarose gel electrophoresis. Thus, heretofore, no method is available to determine the quality of a sample containing RNA in a short time, with a very small quantity, without complication, and with high accuracy.